This proposal aims to bring the new opportunities provided by advances in infrared spectroscopy on the seconds to femtoseconds time scale to fundamental issues remaining in the understanding of biological electron transfer. Although quantum mechanical electron transfer theory has made considerable progress in integrating the broad range of intraprotein electron transfer events from picosecond to seconds and is beginning to offer genuine insights into electron transfer protein engineering, significant questions on the atomic scale remain. The concepts of reorganization energy, protein relaxations and characteristic frequencies of nuclear vibrations coupled to electron transfer are critical to understanding the electron transfers, but knowledge of them remains statistical and devoid of molecular detail. Although the atomic level resolution of the crystal structures of two photosynthetic reactions centers provides a complete static picture of an important electron transfer protein, the structure is silent on important functional events. This proposal describes the means by which direct, functional information on specific bonds of the redox cofactors and their immediate environment can be obtained from infrared spectra of a broad range of well characterized electron transfer reactions that are the foundation of bioenergetics. Experimental design will concentrate on use of extensively studied, highly oriented films with proven native function which have the great advantage of removal of much of bulk water that otherwise obscures infrared measurements. Experiments with both the photosynthetic reaction center and the cytochrome bc1 complex will permit us to explore an entire cycle of electron transfer events in the transformation of redox energy into a chemiosmotic proton and electrical gradient, including the critical events of the coupling of electron transfer to protolytic reactions. Native systems will be studied alongside systems with chemically modified redox cofactors as well as site-directed modification of the protein medium and catalytic sites. We will also introduce an unprecedented flexibility into our IR analysis of redox proteins through the use of de novo construction. Multiple heme and chlorophyll binding synthetic polypeptides, similar to those we have already constructed, will be engineered to simplify the interpretation of IR signals and to address specific questions raised by natural systems. FTIR difference spectra initiated with either light activation or potentiometric redox changes will be extended from the static state down to the microsecond range of kinetics. These observations of the slower electron transfer events will be complemented with state of the art rapid timescale kinetics from nanosecond to pico- and femtoseconds and it will explore protein relaxations and the fastest charge separation events in photosynthesis.